J. H. M. Woltgens

Dentin sialoprotein: biosynthesis and developmental appearance in rat tooth germs in comparison with amelogenins, osteocalcin and colagen type-I

A non-collagenous protein, extracted from rat incisor dentin, is a dentin sialoprotein (DSP). We examined immunohistochemically the developmental appearance and tissue distribution of DSP in 1 to 3-day-old rat molar and incisor tooth germs. The earliest staining for DSP was observed in newly differentiated odontoblasts. In more advanced stages, immunostaining for DSP gradually increased in pre-dentin, odontoblasts and dentin, and appeared in many cells of the dental papilla. In early stages of development before the breakdown of the dental basement membrane, pre-ameloblasts were also positive for DSP. This staining disappeared from the ameloblast cell body soon after deposition of the first layer of mineralized dentin. Radiolabelling of tooth matrix proteins with 14C-serine in vitro followed by immunoprecipitation and fluorography confirmed that DSP was synthesized by tooth-forming cells. The immunolocalization for DSP was different from that of either collagen type-I, osteocalcin or the amelogenins. Whereas collagen type-I and osteocalcin were restricted to the mesenchymal dental tissues, the amelogenins were detectable in both epithelial and mesenchymal dental cells and tissues at the epithelio-mesenchymal interface at early stages of development, prior to the onset of dentin mineralization. We conclude that DSP is expressed in and secreted by odontoblasts and some dental papilla cells from early stages of dentinogenesis onwards, i.e. later than type-I collagen, but before deposition of the first layer of mineralized dentin. In pre-mineralizing stages, some of the matrix proteins may be endocytosed from the pre-dentin by both cell types involved in the epithelio-mesenchymal interaction.

Bone Morphogenetic Protein-7 (Osteogenic Protein-1, OP-1) and Tooth Development

Bone morphogenetic proteins (BMPs) form a family of growth factors originally isolated from extracellular bone matrix that are capable of inducing bone formation ectopically. We studied the expression, tissue localization, and function of BMP-7 (OP-1) during tooth development in rodents. Patterns of BMP-7 gene expression and peptide distribution indicated that BMP-7 was present in dental epithelium during the dental lamina, bud, and cap stages. During the bell stage, BMP-7 mRNA expression and protein distribution shifted from dental epithelium toward the dental mesenchyme. With advancing differentiation of odontoblasts, BMP-7 protein staining in the dental papilla became restricted to the layer of fully functional odontoblasts in the process of depositing (pre)dentin. Secretory-stage ameloblasts exhibited weak immunostaining for BMP-7. A restricted pattern of staining in ameloblasts became apparent in post-secretory stages of amelogenesis. Also, cells of the forming periodontal ligament were immunopositive. Histological analysis of tooth development in neonatal BMP-7-deficient mice did not reveal obvious changes compared with wild-type mice. We conclude that, in developing dental tissues, BMP-7 has distribution and expression patterns similar to those of other BMP members but is not an essential growth factor for tooth development, possibly because of functional redundancy with other BMP members or related growth factors.

Dissimilar Expression Patterns for the Extracellular Matrix Proteins Osteopontin (OPN) and Collagen Type I in Dental Tissues and Alveolar Bone of the Neonatal Rat

Osteopontin (OPN) is a phosphorylated, sialic acid containing glycoprotein that can be extracted from the mineralized extracellular matrix of bone. In the present study we determined the expression patterns of OPN in dental tissues and alveolar bone of 1-3 day old (neonatal) rats by means of 1) immunohistochemistry, 2) Northern blotting and 3) in situ hybridization. We compared these patterns with those of type I collagen. We localized collagen type I expression in osteoblasts adjacent to alveolar bone and in odontoblasts lining predentin/dentin, but not in the epithelial ameloblasts. For OPN, we observed a weak antigenicity in predentin. Although generally no cellular immunostaining was found, very occasionally a minor immunoreactivity was detected in a small number of pre-mineralizing incisor odontoblasts. On the mRNA level, however, no OPN transcripts could be detected in odontoblasts, either by in situ or by Northern hybridization analyses. Also the odontoblasts of the bone-like dentin (osteodentin) region in the tip of incisors were negative for OPN. In contrast, however, osteoblasts of alveolar bone showed strong positive signals with all three techniques, confirming the sensitivity and specificity of the detection methods. From the data obtained in this study, it can be concluded that during early stages of dentinogenesis OPN presumably is not expressed in developing rat tooth germs. The weak immunostaining observed sporadically in some young odontoblasts is probably due to resorption of OPN of non-dental origin entrapped in the predentin.

Use of Fluoride by Young Children and Prevalence of Mottled Enamel

The prevalence of mottled enamel in the permanent dentition of children participating in a fluoride (F-) program at the dental school of the Vrije Universiteit (Amsterdam) was investigated in a study utilizing the Thylstrup-Fejerskov (TF) index. The randomly chosen children received a F- regime considered optimal by the Dutch Advisory Committee for Prevention of Oral and Dental Diseases. From the children examined (n = 83; 49 boys and 34 girls; mean age, 13 years and 5 months), 74% exhibited mottled enamel in a slight to moderate degree. More teeth were affected and the degree of mottling was higher when children started to use F- at an earlier age. Unintentional ingestion of toothpaste containing 0.15% F- during frequent toothbrushing in combination with the daily intake of F- tablets before the age of four may explain the high prevalence of mottled enamel. After these treatments, F- concentrations in plasma of young children can reach values which can directly affect the developing tooth germ.

GENE EXPRESSION AND IMMUNOLOCALISATION OF AMELOGENINS IN DEVELOPING EMBRYONIC AND NEONATAL HAMSTER TEETH

Abstract.Amelogenins are a group of related matrix proteins, synthesised and secreted by ameloblasts during the formation of dental enamel. We have examined expression patterns and the tissue distribution of amelogenins by in situ hybridisation and by immunohistochemistry of developing teeth of embryonic (E12–E15) and neonatal (1- to 4-day-old) golden hamsters. Amelogenin expression and (intracellular) immunostaining for amelogenins were first observed in late embryonic stages in E14 incisors and E15 first molars in partially polarised pre-ameloblasts located along a thin layer of predentine before any overt deposition of enamel. Expression of mRNA and protein staining for amelogenins increased with age and early pre-dentine became immunopositive. The highest mRNA levels and substantial immunostaining for amelogenins were noted in neonatal-stage secretory ameloblasts fully engaged in enamel matrix deposition. After completion of the secretory phase, amelogenin gene expression continued at a lower level in post-secretory stages and was seen in transition-phase and maturation-phase ameloblasts. No amelogenin transcripts were observed in odontoblasts at any stage of their development. However, young odontoblasts stained weakly with anti-amelogenin antibodies before they formed the first layer of dentine, although this staining disappeared in odontoblasts at later stages of development. We conclude that amelogenin gene transcription occurs as early as the polarisation stage of pre-ameloblasts and is closely followed by translation of mRNA into amelogenin proteins. Odontoblasts do not transcribe the amelogenin gene and probably endocytose and digest amelogenins from the pre-dentine. Amelogenins are also transcribed but at a low level in post-secretory stages of amelogenesis.

Fluoride enhances intracellular degradation of amelogenins during secretory phase of amelogenesis of hamster teeth in organ culture

Amelogenins are the major protein species synthesized by secretory ameloblasts and are believed to be involved in enamel mineralization. During enamel formation, amelogenins are progressively degraded into smaller fragments by protease activity. These amelogenin fragments are removed from the enamel extracellular space, thereby enabling full mineralization of the dental enamel. Enamel from fluorotic teeth is porous and contains more proteins and less mineral than sound enamel. In this study we examined the hypothesis that fluoride (F m ) is capable of inhibiting the proteolysis of amelogenins in enamel being formed in organ culture. Hamster molar tooth germs in stages of secretory amelogenesis were pulse labeled in vitro with [ 3 H]- or [ 14 C] proline and subsequently pulse chased. The explants were exposed to F m at different days of chase (i.e., during secretory amelogenesis early after labeling, later after labeling or at stages just beyond secretory amelogenesis). Exposure of secretory stage explants to F m enhanced the release of radiolabeled fragments when F m was applied early after labeling but progressively less if applied later. In contrast, F m had no such effect in stages beyond secretion. The enhanced release of radiolabeled fragments in secretory stages was associated with a reduction of radioactivity in the soft tissue enamel organ indicating that fragmentation of enamel matrix proteins (mainly amelogenins) occurred intracellularly. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the fluorotic enamel contained less radiolabeled parent amelogenins ( M r 28 kD and 26 kD) but more low-molecular-mass fragments than enamel from control explants. Our data indicate that F m promotes intracellular degradation of the newly synthesized parent amelogenins during secretory stage. Our in vitro data do not support the concept that F m impairs extracellular proteolysis of amelogenins, either in the secretory phase or in the stage just beyond the secretory phase.

X-ray micro-analysis of the mineralization patterns in developing enamel in hamster tooth germs exposed to fluoride in vitro during the secretory phase of amelogenesis.

The developing enamel from three-day-old hamster first maxillary (M1) molar tooth germs exposed to fluoride (F-) in vitro was analyzed for its mineral content by means of the energy-dispersive x-ray micro-analysis technique. The aim of this study was to obtain semi-quantitative data on the F--induced hypermineralization patterns in the enamel and to confirm that the increase in electron density observed in micrographs of F-treated enamel (Lyaruu et al., 1986, 1987b) is indeed due to an increase in mineral content in the fluorotic enamel. The tooth germs were explanted during the early stages of secretory amelogenesis and initially cultured for 24 hr in the presence of 10 ppm F- in the culture medium. The germs were then cultured for another 24 hr without F-. In order to compare the ultrastructural results directly with the microprobe data, we used the same specimens for both investigations. The net calcium counts (measurement minus background counts) in the analyses were used as a measure of the mineral content in the enamel. The aprismatic pre-exposure enamel, deposited in vivo before the onset of culture, was the most hypermineralized region in the fluorotic enamel, i.e., it contained the highest amount of calcium measured. The degree of the F--induced hypermineralization gradually decreased (but was not abolished) in the more mature regions of the enamel. The unmineralized enamel matrix secreted during the initial F- treatment in vitro mineralized during the subsequent culture without F-. The calcium content in this enamel layer was in the same order of magnitude as that recorded for the newly deposited enamel in control tooth germs cultured without F-. From these results, and in combination with the ultrastructural data obtained in previous studies, it is concluded that in vitro: (1) F- treatment during the secretory phase of amelogenesis induces hypermineralization of the pre-exposure enamel; (2) F- decontrols or abolishes enamel crystal growth in length and promotes crystal growth in thickness, thus producing enamel hypermineralization; and (3) enamel matrix secreted during F- exposure retains its capacity to support crystal growth when F- is removed from the culture environment.

Influence of Various Inhibitors on Alkaline Phosphatase in Calcifying Hamster Molars, Employing Paranitrophenyl Phosphate as Substrate

In mineralizing hamster molars, the influence of several inhibitors on p-NPP-ase has been demonstrated, and compared with their effect on PPi-ase, known to belong to the same enzyme. Whereas the latter activity is much more physiological, p-NPP is a better substrate to distinguish between the effects of bivalent cations on the enzyme or the substrate.

Effects of Vincristine on the Developing Hamster Tooth Germ in Vitro

Vincristine is one of the cytostatic drugs present in cocktails commonly used for the treatment of cancer in children. The aim of this study was to evaluate biochemically and histologically the toxic effects of this drug on the developing tooth in vitro using the organ culture model in order to be able to predict what damage the drug can induce in the developing teeth from children undergoing anti-neoplastic chemotherapy. The most profound effect of the drug (10(-8)M-10(-4)M vincristine) on the developing tooth germ was the induction of mitotic arrests at the cervical loop and in the inter-cuspal regions. The 10(-4)M-10(-6)M vincristine doses were cytotoxic to most cells in the developing tooth germ. The 10(-7)M vincristine dose apart from induction of mitotic arrests, did not appear to be cytotoxic to the mature differentiated secretory cells. However, this dose induced incomplete nuclear polarization of the differentiating ameloblasts and odontoblasts. At 10(-8)M vincristine, the only effect observed were mitotic arrests; the secretory cells did not appear to have been affected at all. On the other hand, mineralization (TCA-soluble 45Ca and 32P uptake) was dose-dependently decreased from 10(-7)M vincristine upwards. 10(-9)M vincristine, the lowest dose tested, did not induce any changes in the developing tooth germ. The organ culture data indicate that 10(-9)M vincristine is the highest (safe) dose which does not induce any toxic effects in the developing hamster tooth germ.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of colchicine on protein secretion by differentiating odontoblasts and ameloblasts in the hamster tooth in vitro as shown by radioautography with 3H-proline

SummaryWe have examined radioautographically the protein synthetic and secretory activity of differentiating odontoblasts and ameloblasts, exposed for 9 h in vitro to various concentrations of colchicine in the presence of 3H-proline. Colchicine impairs the cytodifferentiation of the dental epithelium into ameloblasts and of the dental mesenchyme into odontoblasts; the effects depend on the dose. However, denial epithelial cells are more sensitive to the drug than dental mesenchymal cells. In stages prior to odontoblast differentiation, colchicine enhances the number of radioautographic grains over the dental epithelium without changing the grain counts over the dental basement membrane area: This suggests that in vitro the dental epithelium synthesizes and secretes proline-containing components that are not constituents of the dental basement membrane. Also, during the subsequent stages of ameloblast differentiation colchicine increases the number of radioautographic grains over the preameloblasts. The present data suggest that the primary in vitro target of colchicine is not the dental mesenchyme, but the dental epithelium. The data also indicate that differentiating ameloblasts synthesize and secrete significant amounts of proteins in vitro prior to the first deposition of enamel.